Generating primary mouse models of diffuse midline glioma through a combination of the RCAS/tv- a retrovirus system and CRISPR/Cas9 gene editing

Abstract Diffuse midline gliomas (DMGs) are deadly brain tumors that arise in the brainstem of children and young adults, resulting in a median survival of less than two years. Genetically engineered mouse models (GEMMs) are critical to studying tumorigenesis and tumor-immune interactions in DMG, which may inform the design of new urgently-needed treatment approaches. One approach to generate DMG GEMMs uses RCAS/tv-a, an avian retrovirus gene delivery system to express transgenes in specific brain cell lineages in the mouse brain. However, this approach often requires resource-intensive breeding to generate mice with the desired genetic alterations in their germline. As a result, we aimed to establish a new approach that combines this system with CRISPR/Cas9 gene editing technology. This methodology has been described for model adult supratentorial gliomas in mice but has not been described for pediatric or brainstem gliomas. To express Cas9 and the avian retrovirus receptor TVA in mouse neural stem cells, we bred mice of genotype Nestin-TVA; Nestin-Cre; LoxP-STOP-LoxP-Cas9-EGFP. RCAS retrovirus plasmids were generated containing various guide RNAs (gRNAs) targeting DMG-relevant genes for disruption by Cas9, including Trp53, Cdkn2a, Atm, Ppm1d, Pten, and a non-targeting control (Cntl). For each gRNA, two plasmids were generated, one with a platelet-derived growth factor beta ligand (PDGFB) oncogene driver and one with a blue fluorescent protein (BFP) marker. In a pilot experiment, avian fibroblast cells transduced with RCAS-Trp53_gRNA_BFP and RCAS-Cntl_gRNA-PDGFB were injected into postnatal day 4 mouse brainstems of the aforementioned genotype and observed for tumor formation. High grade tumor formation was observed. We also observed high grade tumors in mice of genotype Nestin-TVA; LoxP-STOP-LoxP-Cas9-EGFP when injected with an additional RCAS-Cre construct, indicating that Cre can be expressed from the mouse germline or delivered via a retrovirus payload to drive tumorigenesis. Immunohistochemistry confirmed tumor features that model human DMG, expression of Cas9 and PDGFB, and loss of p53. Thus, RCAS/tv-a and CRISPR/Cas9 can be combined to model pediatric glioma tumorigenesis in the mouse brainstem. This approach to mouse modeling will streamline future GEMM DMG research by allowing new genes of interest to be rapidly perturbed by simply generating RCAS retrovirus constructs. The workflow may also enable sophisticated experiments such as pooled in vivo genetic experiments that screen gRNAs targeting various tumor suppressor genes or therapeutic targets. Citation Format: Sophie R. Wu, Harrison Q. Liu, Joshua Tolliver, Spencer M. Maingi, Vennesa Valentine, Nerissa T. Williams, Zachary J. Reitman. Generating primary mouse models of diffuse midline glioma through a combination of the RCAS/tv-a retrovirus system and CRISPR/Cas9 gene editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1437.